Measurement of circulating tumor cells (CTC) has potential to fill major gaps in the clinical care and management of lung cancer patients. Validation of CTC as a prognostic marker in breast cancer patients set an important precedent for CTC measurement; however, histochemical CTC enumeration now used as a clinical correlate in breast and prostate cancer has proven to be neither robust nor reliable enough for application to lung cancer. This is due in large part to the phenotypic heterogeneity and high antigen variability associated with lung tumors as compared to other solid tumors. PCR amplification of cancer protein transcripts is a conceptually appealing alternative approach to CTC measurement that offers sensitivity and unlimited capabilities for multiple antigen measurements. Although literature indicates that PCR detection of CTC correlates with lung cancer stage, disease recurrence and survival, this approach remains investigational. Since mRNA transcript is used as a surrogate for tumor protein expression, PCR is criticized for lack of specificity. We have devised an approach that combines the specificity of immuno-histochemistry with the sensitivity of PCR. This application details testing and optimization of a novel nanoparticle-based system to overcome technical and biological hurdles encountered in lung cancer CTC measurement. Specifically, bio-nanoconjugates are constructed to carry one antigen specific monoclonal antibody and a conjugate-specific double stranded DNA tag sequence for nano-specific identification. When conjugates bind antigen on a cell surface, a single strand of the DNA tag can be released into suspension by briefly heating the complex mixture; liberated DNA strands amplified by PCR provide a highly sensitive and quantifiable measure of cellular binding and corresponding antigen expression. Signal amplification of the antigen-bound bio-nanoconjugate allows detection of low frequency proteins on low concentration CTC in peripheral blood, and multiplexing capabilities inherent to the technology offer increasing sensitivity without reduced specificity, making this particularly well suited for the measurement of lung cancer CTC. Sensitivity of the approach will be determined with peripheral blood spiked at various concentrations with cultured tumor cells with known antigen expression. CTC enrichment using antibody-mediated separation of CTC from peripheral blood mononuclear cells is expected to further increase the limits of detection, while also reducing the potential for false positive results. We anticipate being able to achieve unrivaled detection limits with high degrees of specificity for lung cancer CTC. This application details the comparative and systematic phases of initial development and testing of this promising initiative.